Bioelectronic interfaces that achieve electrical communication between redox enzymes and an electrode have applications as biosensors (Armstrong et al., 1997, Halbhuber et al., 2003, Zayats et al., 2002), biocatalysts (Park et al., 1999, Park et al., 2003, Park and Zeikus, 1999, Tsujimura et al., 2001), and biofuel cells (Chen et al., 2001, Park and Zeikus, 2003). Development of bioelectronic interfaces is especially challenging for dehydrogenase enzymes, whose activity requires the presence of an electron carrying cofactor [e.g., β-nicotinamide adenine dinucleotide (phosphate) (NAD(P)+)] in the Rossmann fold of the enzyme. The cofactor facilitates the transfer of electrons between the redox center of the enzyme and the electrode. However, direct electrochemical oxidation of NADH requires the use of high overpotentials, which may lead to cofactor degradation (Blaedel and Jenkins, 1975, Schmakel et al., 1975). Cofactor degradation can be circumvented using an electron mediator, such as toluidine blue O (TBO), Nile blue A, or neutral red to shuttle electrons between the electrode and cofactor at moderate potentials (Molina et al., 1999, Pasco et al., 1999).
Several approaches have been used to achieve mediated electron exchange, including the development of linear (Zayats et al., 2002, Hassler et al., 2007) and branched (Hassler et al., 2007, Hassler and Worden, 2006) molecular architectures that simultaneously hold the electrode, mediator, cofactor, and enzyme in close proximity, allow unimpeded access of the cofactor to its binding site on the enzyme, provide efficient, multistep electron transfer, and prevent component loss due to diffusion. However, these fabrication methods involve covalent linkages and make no provision for removal and replacement of labile components, such as the enzyme and cofactor, which have limited lifetimes. Long-term operation requires interface assembly methods that allow periodic removal and replacement of these components.
A method to fabricate renewable bioelectronic interface on gold electrodes (Hassler et al., 2007) has been developed. This method allows facile removal and replacement of the cofactor and enzyme. The approach uses layer-by-layer deposition of polyelectrolytes to reversibly bind the cofactor and enzyme, so that they can be removed by reducing pH and then replaced to regenerate the bioelectronic activity (Hassler et al., 2007).
However, because this method uses a thiol linkage to anchor the interface to the gold electrode, it may not be suitable for other electrode materials. In addition, thiol bonds may have disadvantages for certain applications. Alkanethiols tend to desorb at potentials outside the potential window defined by 800 to −1400 mV (vs Ag/AgCl) (Walczak et al., 1991, Widrig et al., 1991) and at temperatures over 100° C. (Bhatia and Garrison, 1997). Also, the gold/thiol junction generates a significant tunneling barrier (−2 eV) (Ranganathan et al., 2001). Alkoxy-terminated silanes can react with surface hydroxyl groups on metal-oxide electrodes to form a polysiloxane linkage (Curran et al., 2005, Quan et al., 2004). However, Kraft has reported that metal oxide substrates are not stable during anodic potential cycling, due to the anodic dissolution of the metal-oxide coating (Kraft et al., 1994).